Lenvatinib is an orally effective tyrosine kinase inhibitor used to treat several types of tumors, including progressive, radioiodine-refractory differentiated thyroid cancer and advanced renal cell carcinoma. Although this drug is increasingly used in therapy, its metabolism and effects on the organism are still not described in detail. Using the rat as an experimental animal model, this study aimed to investigate the metabolism of lenvatinib by rat microsomal enzymes and cytochrome P450 (CYPs) enzymes recombinantly expressed in SupersomesTMin vitro and to assess the effect of lenvatinib on rat CYP expression in vivo. Two metabolites, O-desmethyl lenvatinib, and lenvatinib N-oxide, were produced by rat CYPs in vitro. CYP2A1 and 2C12 were found to be the most effective in forming O-desmethyl lenvatinib, while CYP3A2 was found to primarily form lenvatinib N-oxide. The administration of lenvatinib to rats caused changes in the expression of mRNA and protein, as well as the activity of various CYPs, particularly in an increase in CYP1A1. Thus, the administration of lenvatinib to rats has an impact on the level of CYPs.
- MeSH
- chinoliny * farmakologie MeSH
- fenylmočovinové sloučeniny * farmakologie MeSH
- inhibitory proteinkinas * farmakologie MeSH
- inhibitory tyrosinkinasy MeSH
- jaterní mikrozomy účinky léků MeSH
- játra * účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- messenger RNA metabolismus genetika MeSH
- oxidace-redukce * účinky léků MeSH
- potkani Sprague-Dawley MeSH
- systém (enzymů) cytochromů P-450 * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Endocrine disruptors (EDs) are compounds that interfere with the balance of the endocrine system by mimicking or antagonising the effects of endogenous hormones, by altering the synthesis and metabolism of natural hormones, or by modifying hormone receptor levels. The synthetic estrogen 17α-ethinylestradiol (EE2) and the environmental carcinogen benzo[a]pyrene (BaP) are exogenous EDs whereas the estrogenic hormone 17β-estradiol is a natural endogenous ED. Although the biological effects of these individual EDs have partially been studied previously, their toxicity when acting in combination has not yet been investigated. Here we treated Wistar rats with BaP, EE2 and estradiol alone or in combination and studied the influence of EE2 and estradiol on: (i) the expression of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver on the transcriptional and translational levels; (ii) the inducibility of these CYP enzymes by BaP in this rat organ; (iii) the formation of BaP-DNA adducts in rat liver in vivo; and (iv) the generation of BaP-induced DNA adducts after activation of BaP with hepatic microsomes of rats exposed to BaP, EE2 and estradiol and with recombinant rat CYP1A1 in vitro. BaP acted as a strong and moderate inducer of CYP1A1 and 1B1 in rat liver, respectively, whereas EE2 or estradiol alone had no effect on the expression of these enzymes. However, when EE2 was administered to rats together with BaP, it significantly decreased the potency of BaP to induce CYP1A1 and 1B1 gene expression. For EE2, but not estradiol, this also correlated with a reduction of BaP-induced CYP1A1 enzyme activity in rat hepatic microsomes. Further, while EE2 and estradiol did not form covalent adducts with DNA, they affected BaP-derived DNA adduct formations in vivo and in vitro. The observed decrease in BaP-DNA adduct levels in rat liver in vivo resulted from the inhibition of CYP1A1-mediated BaP bioactivation by EE2 and estradiol. Our results indicate that BaP genotoxicity mediated through its activation by CYP1A1 in rats in vivo is modulated by estradiol and its synthetic derivative EE2.
- MeSH
- benzopyren toxicita MeSH
- cytochrom P-450 CYP1A1 biosyntéza genetika MeSH
- endokrinní disruptory toxicita MeSH
- estradiol toxicita MeSH
- ethinylestradiol toxicita MeSH
- jaterní mikrozomy účinky léků enzymologie MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- regulace genové exprese enzymů * účinky léků MeSH
- synergismus léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
OBJECTIVES: The term "endocrine disruptor" (ED) is used for compounds that mimic or antagonize the effects of endogenous hormones. Synthetic estrogen 17α-ethinylestradiol (EE2) and a human carcinogen benzo[a]pyrene (BaP) are assigned as exogenous endocrine disruptors and an estrogenic hormone estradiol is a natural endogenous disruptor. Here, the potency of these three disruptors administered to rats individually and in combination to induce expression of cytochrome P450 (CYP) enzymes involved in their own metabolism (CYP1A1, 2C and 3A) in vivo was investigated. METHODS: Changes in CYP protein expression after exposure of rats to BaP, EE2 or estradiol were analyzed by Western blotting. Using the HPLC method, CYP1A1, 2C and 3A specific activities in hepatic microsomes isolated from exposed rats were analyzed. RESULTS: Whereas exposure to BaP induces expression of CYP1A1 protein and its marker activity (Sudan I oxidation) in liver, kidney and lung of rats, no significant induction of this CYP and its enzyme activity was produced by EE2 and estradiol. Treatment of BaP in combination with EE2 and/or estradiol decreased the BaP-mediated CYP1A1 induction in liver of exposed rats. BaP also induces CYP2C11 protein in rat liver and kidney, but does not increase its enzyme activity measured as testosterone 16α-hydroxylation. The enzyme activity of another enzyme of the 2C subfamily, CYP2C6, diclofenac 4'-hydroxylation, is even decreased by BaP. The CYP2C11 protein expression and/or its activity are also increased in liver of rats treated with EE2 and estradiol, but its expression is significantly decreased in lung. The CYP2C6 activity is also elevated by treatment of rats with EE2 and estradiol administered individually as well as in their combination. Whereas only a slight increase in CYP3A protein expression was found by BaP in rat liver, its enzyme activity, testosterone 6β-hydroxyalation, increased significantly in this organ. In contrast, no effect or even a decrease in CYP3A expression and its enzyme activity was produced by EE2 and estradiol in rats exposed to these compounds.
- MeSH
- benzopyren farmakologie MeSH
- endokrinní disruptory farmakologie MeSH
- estradiol farmakologie MeSH
- estrogeny farmakologie MeSH
- ethinylestradiol farmakologie MeSH
- jaterní mikrozomy metabolismus MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
UNLABELLED: Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H: quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the (32)P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.
- MeSH
- adukty DNA antagonisté a inhibitory biosyntéza MeSH
- cytochrom P-450 CYP1A1 biosyntéza MeSH
- cytochrom P-450 CYP1A2 biosyntéza MeSH
- enzymová indukce účinky léků fyziologie MeSH
- karcinogeny toxicita MeSH
- krysa rodu rattus MeSH
- kyseliny aristolochové toxicita MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein-protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein-protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes and to identify the crucial portions of the interacting molecules. This study is devoted to structure-functional relationships in the microsomal Mixed Function Oxidase (MFO) complex, which is responsible for biotransformation of many hydrophobic endogenous compounds and xenobiotics. In particular, the cytochrome b5 interaction with MFO terminal oxygenase cytochrome P-450 (P450) was studied. To create photolabile probes suitable for this purpose, we prepared cytochrome b5 which had a photolabile diazirine analog of methionine (pMet) incorporated into the protein sequence, employing recombinant expression in Escherichia coli. In addition to wild-type cytochrome b5, where three methionines (Met) are located at positions 96, 126, and 131, six mutants containing only one Met in the sequence were designed and expressed (see Table 1). In these mutants, a single Met was engineered into the catalytic domain (at positions 23, 41, or 46), into the linker between the protein domains (at position 96), or into the membrane region (at positions 126 or 131). These mutants should confirm or exclude these portions of cytochrome b5 which are involved in the interaction with P450. After UV irradiation, the pMet group(s) in the photolabile cytochrome b5 probe was(were) activated, producing covalent crosslinks with the interacting parts of P450 2B4 in the close vicinity. The covalent complexes were analyzed by the "bottom up" approach with high-accuracy mass spectrometry. The analysis provided an identification of the contacts in the supramolecular complex with low structural resolution. We found that all the above-mentioned cytochrome b5 Met residues can form intermolecular crosslinks and thus participate in the interaction. In addition, our results indicate the existence of at least two P450:cytochrome b5 complexes which differ in the orientation of individual proteins. The results demonstrate the advantages of the photo-initiated crosslinking technique which is able to map the protein-protein interfaces not only in the solvent exposed regions, but also in the membrane-embedded segments (compared to a typical crosslinking approach which generally only identifies crosslinks in solvent exposed regions).
- MeSH
- aromatické hydroxylasy analýza chemie metabolismus MeSH
- cytochromy b5 analýza chemie metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- králíci MeSH
- mapy interakcí proteinů fyziologie MeSH
- reagencia zkříženě vázaná chemie metabolismus MeSH
- světelná stimulace metody MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
